Manipulation of organic acid biosynthesis and secretion

ABSTRACT

The present invention relates to nucleic acid fragments encoding amino acid sequences for organic acid biosynthetic enzymes in plants, and the use thereof for the modification of, for example, organic acid biosynthesis and secretion in plants. In particularly preferred embodiments, the invention relates to the combinatorial expression of citrate synthase (CS) and/or malate dehydrogenase (MDH) and/or phosphoenolpyruvate carboxylase (PEPC) in plants to modify, for example, organic acid synthesis and secretion.

The present invention relates to nucleic acid fragments encoding aminoacid sequences for organic acid biosynthetic enzyme polypeptides inplants, and the use thereof for the modification of organic acidbiosynthesis and secretion in plants. In particularly preferredembodiments, the invention relates to the combinatorial expression ofmalate dehydrogenase (MDH) and/or phosphoenolpyruvate carboxylase (PEPC)and/or citrate synthase (CS) in plants to modify organic acidbiosynthesis and secretion.

Documents cited in this specification are for reference purposes onlyand their inclusion is not acknowledgment that they form part of thecommon general knowledge in the relevant art.

Organic acids, such as citrate and malate, are key metabolites inplants. They are involved in numerous processes, including C4 andCrassulacean acid metabolism (CAM) photosynthesis, stomatal andpulvinual movement, nutrient uptake, respiration, nitrogen assimilation,fatty acid oxidation, and providing energy to bacteroids in rootnodules. For example, malate plays a key role in root nodule metabolismand nitrogen fixation, serving as the primary carbon source forbacteroid maintenance and nitrogenase activity, and is also tightlylinked to nodule nitrogen assimilation. Furthermore, the complexing roleof organic acids produced and excreted from plant roots has also beenassociated with tolerance to the aluminium cation Al³ ⁺which is toxic tomany plants at micromolar concentrations. Aluminium toxicity has beenrecognized as a major limiting factor of plant productivity on acidicsoils, which account for approximately 40% of the earth's arable land.

The tricarboxylic acid cycle (TCA), also known as Krebs cycle (after itsdiscoverer Hans Krebs) or citric acid cycle, moves electrons fromorganic acids to the oxidized redox cofactors NAD⁺and FAD, forming NADH,FADH₂, and carbon dioxide (CO₂). The reaction sequence of the TCA cycleinvolves: in a reaction catalysed by citrate synthase (CS), acetyl-CoAformed by the pyruvate dehydrogenase complex combines with oxaloacetateto produce the C₆ tricarboxylic acid, citrate. In the overall cycle, thecitrate is oxidized to produce two molecules of CO₂ in a series ofreactions that leads to the formation of one oxaloacetate, three NADH,one FADH₂, and one ATP. The resulting oxaloacetate reacts with anothermolecule of acetyl-CoA to continue the cycle. The oxidativedecarboxylation of pyruvate yields an additional CO₂ and NADH. Thus theTCA cycle brings about the complete oxidation of pyruvate to three CO₂plus 10 electrons, which are stored temporarily as 4 NADH and 1 FADH₂.

Cytosolic reactions generate products that are transported into themitochondria to feed the TCA cycle. The nature of the end product of theglycolytic reactions in the cytosol of plants is determined by therelative activities of the three enzymes that can utilizephosphoenol-pyruvate (PEP) as substrate. Both pyruvate kinase andPEP-phosphatase form pyruvate; while PEP-carboxylase (PEPC) generatesoxaloacetate. Pyruvate is transported directly into the mitochondrion.Oxaloacetate is either transported directly into the mitochondrion orfirst reduced to malate by cytosolic malate dehydrogenase (MDH).

Before entering the TCA cycle proper, pyruvate is oxidised anddecarboxylated by the pyruvate dehydrogenase enzyme complex to form CO₂,acetyl-CoA, and NADH. The pyruvate dehydrogenase enzyme complex, whichrequires the bound cofactors thiamine pyrophosphate, lipoic acid, andFAD as well as free coenzyme A (CoASH) and NAD+, links the TCA cycle toglycolysis.

It is known that the TCA cycle includes the following enzymes: pyruvatedehydrogenase, citrate synthase, citrate hydrolase, isocitratedehydrogenase, oxoglutarate dehydrogenase, succinyl-CoA synthetase,succinate dehydrogenase, fumarase, malate dehydrogenase, NAD-malicenzyme and phosphoenolpyruvate carboxylase.

In particular, citrate synthase (CS) catalyses the condensation ofacetyl-CoA and oxaloacetate to form the C6 molecule citrate and freeCoASH, as the TCA cycle proper begins.

Malate dehydrogenase (MDH) catalyses the final step of the TCA cycle,oxidizing malate to oxaloacetate and producing NADH. This reactioncatalysed by MDH is reversible, thus allowing also for the reversiblereduction of oxaloacetate to malate. The enzyme MDH is important inseveral metabolic pathways, and higher plants contain multiple formsthat differ in co-enzyme specificity and subcellular localization.Chloroplasts contain an NADP⁺-dependent MDH that plays a critical rolein balancing reducing equivalents between the cytosol and stroma. Plantsalso contain NAD-dependent MDHs which are found in a) mitochondria aspart of the TCA cycle; b) cytosol and peroxisomes involved inmalate-aspartate shuttles; and c) glyoxisomes functioning inβ-oxidation. In root nodules of nitrogen-fixing legumes, such as whiteclover (Trifolium repens) and alfalfa (Medicago sativa), malate servesas the primary carbon source to support the respiratory needs of thebacterial microsymbiont and the fixation of N₂ by nitrogenase, and anodule-enhanced MDH is thus critical for nodule function.

Phosphoenolpyruvate carboxylase (PEPC) catalyses the reaction ofphosphoenol-pyruvate with HCO₃ ⁻releasing the phosphate and producingthe C4 product, oxaloacetate. Oxaloacetate is commonly reduced to malateby NADH through the action of malate dehydrogenase (MDH). PEPC is ahomotetrameric enzyme widely distributed in most plant tissues. Inplants, PEPC fulfills various physiological roles such as thephotosynthetic CO₂ fixation in C₄ and Crassulacean Acid Metabolism (CAM)plants, and the anaplerotic pathway.

While nucleic acid sequences encoding some organic acid biosyntheticenzymes have been isolated for certain species of plants, there remainsa need for materials useful in modifying organic acid biosynthesis; inmodifying organic acid secretion; in modifying phosphorus acquisitionefficiency in plants; in modifying aluminium and acid soil tolerance inplants; in modifying nitrogen fixation and nodule function, particularlyin forage legumes and grasses, including alfalfa, medics, clovers,ryegrasses and fescues, and for methods for their use.

This invention is directed towards overcoming, or at least alleviating,one or more of the difficulties or deficiencies associated with theprior art.

In one aspect, the present invention provides substantially purified orisolated nucleic acids or nucleic acid fragments encoding the organicacid biosynthetic polypeptides CS, MDH and PEPC, from a clover(Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca)species, or functionally active fragments or variants of thesepolypeptides.

The present invention also provides substantially purified or isolatednucleic acids or nucleic acid fragments encoding amino acid sequencesfor a class of polypeptides which are related to CS, MDH and PEPC (froma clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue(Festuca) species) of CS, MDH and PEPC, or functionally active fragmentsor variants of CS, MDH and PEPC. Such polypeptides are referred toherein as CS-like, MDH-like and PEPC-like respectively and includepolypeptides having similar functional activity.

The present invention also relates to individual or simultaneousenhancement or otherwise manipulation of CS, MDH and/or PEPC or likegene activities in plants to enhance or otherwise alter organic acidbiosynthesis; to enhance or reduce or otherwise alter organic acidsecretion; to enhance or reduce or otherwise alter phosphorousacquisition efficiency in plants; to enhance or reduce or otherwisealter aluminium and acid soil tolerance in plants; and/or to enhance orreduce or otherwise alter nitrogen fixation and nodule function inlegumes.

The individual or simultaneous enhancement or otherwise manipulation ofCS, MDH and/or PEPC or like gene activities in plants has significantconsequences for a range of applications in, for example, plantproduction, plant performance, plant nutrition and plant tolerance. Forexample, it has applications in increasing plant tolerance toaluminium-toxic acid soils; in improving plant nutrient acquisitionefficiency for example in increasing acquisition of phosphorus fromsoils; in increasing nodule function in nitrogen-fixing legumes forexample leading to enhanced nitrogen fixation; in modifying theaccumulation of organic acids such as citrate in fruits; in modifyingthe secretion of organic acids for example citrate and/or malate fromplant roots.

Manipulation of CS, MDH and/or PEPC or like gene activities in plants,including legumes such as clovers (Trifolium species), lucerne (Medicagosativa) and grass species such as ryegrasses (Lolium species) andfescues (Festuca species) may be used to facilitate the production of,for example, forage legumes and forage grasses and other crops withenhanced tolerance to aluminium toxic soils; enhanced nutrientacquisition efficiency; forage legumes with enhanced nitrogen fixation;fruits with enhanced organic acid content leading to enhanced flavourand health benefits.

The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue(Festuca) species may be of any suitable type, including white clover(Trifolium repens), red clover (Trifolium pratense), subterranean clover(Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annualryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tallfescue (Festuca arundinacea), meadow fescue (Festuca pratelisis) and redfescue (Festuca rubra). Preferably the species is a clover or aryegrass, more preferably white clover (T. repens) or perennial ryegrass(L. perenne). White clover (Trifolium repens L.) and perennial ryegrass(Lolium perenne L.) are key pasture legumes and grasses, respectively,in temperate climates throughout the world. Perennial ryegrass is alsoan important turf grass.

The nucleic acid or nucleic acid fragment may be of any suitable typeand includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA)that is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases, and combinations thereof. TheRNA is readily obtainable, for example, by transcription of a DNAsequence according to the present invention, to produce a RNAcorresponding to the DNA sequence. The RNA may be synthesised in vivo orin vitro or by chemical synthesis to produce a sequence corresponding toa DNA sequence by methods well known in the art. In this specification,where the degree of sequence similarity between an RNA and DNA is suchthat the strand of the DNA could encode the RNA, then the RNA is said to“correspond” to that DNA.

The term “isolated” means that the material is removed from its originalenvironment (eg. the natural environment if it is naturally occurring).For example, a naturally occurring nucleic acid or polypeptide presentin a living plant is not isolated, but the same nucleic acid orpolypeptide separated from some or all of the coexisting materials inthe natural system, is isolated. Such an isolated nucleic acid could bepart of a vector and/or such a nucleic acid could be part of acomposition, and still be isolated in that such a vector or compositionis not part of its natural environment. An isolated polypeptide could bepart of a composition and still be isolated in that such a compositionis not part of its natural environment.

By “functionally active” in respect of a nucleic acid it is meant thatthe fragment or variant is capable of modifying organic acidbiosynthesis in a plant. A variant in this context can be an analogue,derivative or mutant and includes naturally occurring allelic variantsand non-naturally occurring variants. Additions, deletions,substitutions and derivatizations of one or more of the nucleotides arecontemplated so long as the modifications do not result in loss offunctional activity of the fragment or variant. Preferably thefunctionally active fragment or variant has at least approximately 80%identity to the functional part of the above mentioned sequence, morepreferably at least approximately 90% identity, most preferably at leastapproximately 95% identity. Such functionally active variants andfragments include, for example, those having nucleic acid changes whichresult in conservative amino acid substitutions of one or more residuesin the corresponding amino acid sequence. Preferably the fragment has asize of at least 30 nucleotides, more preferably at least 45nucleotides, most preferably at least 60 nucleotides.

By “functionally active” in respect of a polypeptide it is meant thatthe fragment or variant has one or more of the biological properties ofthe proteins CS, CS-like, MDH,

MDH-like, PEPC and PEPC-like. A variant in this context includesadditions, deletions, substitutions and derivatizations of one or moreof the amino acids are contemplated so long as the modifications do notresult in loss of functional activity of the fragment or variant.Preferably the functionally active fragment or variant has at leastapproximately 60% identity to the functional part of the above mentionedsequence, more preferably at least approximately 80% identity, mostpreferably at least approximately 90% identity.

Such functionally active variants and fragments include, for example,those having conservative amino acid substitutions of one or moreresidues in the corresponding amino acid sequence. Preferably thefragment has a size of at least 10 amino acids, more preferably at least15 amino acids, most preferably at least 20 amino acids.

The term “construct” as used herein refers to an artificially assembledor isolated nucleic acid molecule which includes the gene of interest.In general a construct may include the gene or genes of interest, amarker gene which in some cases can also be the gene of interest andappropriate regulatory sequences. It should be appreciated that theinclusion of regulatory sequences in a construct is optional, forexample, such sequences may not be required in situations where theregulatory sequences of a host cell are to be used. The term constructincludes vectors but should not be seen as being limited thereto.

The term “vector” as used herein encompasses both cloning and expressionvectors. Vectors are often recombinant molecules containing nucleic acidmolecules from several sources.

By “operatively linked” in respect of a regulatory element, nucleic acidor nucleic acid fragment and terminator, is meant that the regulatoryelement is capable of causing expression of said nucleic acid or nucleicacid fragment in a plant cell and said terminator is capable ofterminating expression of said nucleic acid or nucleic acid fragment ina plant cell. Preferably, said regulatory element is upstream of saidnucleic acid or nucleic acid fragment and said terminator is downstreamof said nucleic acid or nucleic acid fragment.

By “an effective amount” of a nucleic acid or nucleic acid fragment ismeant an amount sufficient to result in an identifiable phenotypic traitin said plant, or a plant, plant seed or other plant part derivedtherefrom. Such amounts can be readily determined by an appropriatelyskilled person, taking into account the type of plant, the route ofadministration and other relevant factors. Such a person will readily beable to determine a suitable amount and method of administration. See,for example, Maniatis et al, Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, the entire disclosure of which is incorporated herein byreference.

It will also be understood that the term “comprises” (or its grammaticalvariants) as used in this specification is equivalent to the term“includes” and should not be taken as excluding the presence of otherelements or features.

Such nucleic acids or nucleic acid fragments could be assembled to forma consensus contig. As used herein, the term “consensus contig” refersto a nucleotide sequence that is assembled from two or more constituentnucleotide sequences that share common or overlapping regions ofsequence homology. For example, the nucleotide sequence of two or morenucleic acids or nucleic acid fragments can be compared and aligned inorder to identify common or overlapping sequences. Where common oroverlapping sequences exist between two or more nucleic acids or nucleicacid fragments, the sequences (and thus their corresponding nucleicacids or nucleic acid fragments) can be assembled into a singlecontiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencodes a CS or CS-like polypeptide and including a nucleotide sequenceselected from the group consisting of (a) sequences shown in SEQ ID NOS1, 3 to 10, 11, 13 to 16, 17, 19, 327, 329 to 335, 336, 338 to 344, 349,351 and 353; (b) complements of the sequences recited in (a); (c)sequences antisense to the sequences recited in (a) and (b); (d)functionally active fragments and variants of the sequences recited in(a), (b) and (c); and (e) RNA sequences corresponding to the sequencesrecited in (a), (b), (c) and (d).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencodes a MDH or MDH-like polypeptide and including a nucleotidesequence selected from the group consisting of (a) sequence shown in SEQID NOS. 21, 23 to 29; 30, 32 to 33, 34, 36, 38, 40, 42 to 43, 44, 46, 48to 110, 111, 113, 115, 117 to 182, 183, 185, 205, 207 to 217, 218, 220to 251, 252, 254 to 270, 271, 273 to 275, 276, 278 to 287, 288, 290 to292, 293, 295 to 296, 297, 299 to 301, 304 to 305, 306 and 308; (b)complements of the sequences recited in (a); (c) sequences antisense tothe sequences recited in (a) and (b); (d) functionally active fragmentsand variants of the sequences recited in (a), (b) and (c); and (e) RNAsequences corresponding to the sequences recited in (a), (b), (c) and(d).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencodes a PEPC or PEPC-like polypeptide and including a nucleotidesequence selected from the group consisting of (a) sequences shown inSEQ ID NOS 187, 189, 191 to 197, 199, 201, 203, 310, 312 to 314, 315,317 to 318, 319, 321 to 322, 323, 325 and 347; (b) complements of thesequences recited in (a); (c) sequences antisense to the sequencesrecited in (a) and (b); (d) functionally active fragments and variantsof the sequences recited in (a), (b) and (c); and (e) RNA sequencescorresponding to the sequences recited in (a), (b), (c) and (d).

Nucleic acids or nucleic acid fragments encoding at least a portion ofseveral CS, MDH and PEPC polypeptides have been isolated and identified.Genes encoding other CS or CS-like, MDH or MDH-like and PEPC orPEPC-like proteins, either as cDNAs or genomic DNAs, may be isolateddirectly by using all or a portion of the nucleic acids or nucleic acidfragments of the present invention as hybridisation probes to screenlibraries from the desired plant employing the methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon thenucleic acid sequences of the present invention may be designed andsynthesized by methods known in the art. Moreover, the entire sequencesmay be used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labelling, nick translation,or end-labelling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers may be designed andused to amplify a part or all of the sequences of the present invention.The resulting amplification products may be labelled directly duringamplification reactions or labelled after amplification reactions, andused as probes to isolate full-length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acidfragments of the present invention may be used in protocols to amplifylonger nucleic acids or nucleic acid fragments encoding homologous genesfrom DNA or RNA. For example, polymerase chain reaction may be performedon a library of cloned nucleic acid fragments wherein the sequence ofone primer is derived from the nucleic acid sequences of the presentinvention, and the sequence of the other primer takes advantage of thepresence of the polyadenylic acid tracts to the 3′ end of the mRNAprecursor encoding plant genes. Alternatively, the second primersequence may be based upon sequences derived from the cloning vector.For example, those skilled in the art can follow the RACE protocol(Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998, the entiredisclosure of which is incorporated herein by reference) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3′ or 5′ end. Using commerciallyavailable 3′ RACE and 5′ RACE systems (BRL), specific 3′ or 5′ cDNAfragments may be isolated (Ohara et al. (1989) Proc. Natl. Acad Sci USA86:5673; Loh et al. (1989) Science 243:217, the entire disclosures ofwhich are incorporated herein by reference). Products generated by the3′ and 5′ RACE procedures may be combined to generate full-length cDNAs.

In a further aspect of the present invention there is provided asubstantially purified or isolated polypeptide from a clover(Trifolium), medic (Medicago), ryegrass (Loliuni) or fescue (Festuca)species, selected from the group consisting of CS or CS-like, MDH orMDH-like and PEPC or PEPC-like polypeptides; and functionally activefragments and variants of these polypeptides.

The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue(Festuca) species may be of any suitable type, including white clover(Trifolium repens), red clover (Trifolium pratense), subterranean clover(Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annualryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tallfescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and redfescue (Festuca rubra). Preferably the species is a clover or aryegrass, more preferably white clover (T. repens) or perennial ryegrass(L. perenne).

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated CS or CS-like polypeptide includes anamino acid sequence selected from the group consisting of sequencesshown in SEQ ID NOS 2, 12, 18, 20, 328, 337, 350, 352 and 354; andfunctionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated MDH or MDH-like polypeptide includesan amino acid sequence selected from the group consisting of sequencesshown in SEQ ID NOS 22, 31, 35, 37, 39, 41, 45, 47, 112, 114, 116, 184,186, 206, 219, 253, 272, 277, 289, 294, 297, 303, 307 and 309; andfunctionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated PEPC or PEPC-like polypeptideincludes an amino acid sequence selected from the group consisting ofsequences shown in SEQ ID NOS 188, 190, 198, 200, 202, 204, 311, 316,320, 324, 326, and 348; and functionally active fragments and variantsthereof.

In a further embodiment of this aspect of the invention, there isprovided a polypeptide produced (e.g. recombinantly) from a nucleic acidor nucleic acid fragment according to the present invention. Techniquesfor recombinantly producing polypeptides are known to those skilled inthe art.

Availability of the nucleotide sequences of the present invention anddeduced amino acid sequences facilitates immunological screening of cDNAexpression libraries. Synthetic peptides representing portions of theinstant amino acid sequences may be synthesized. These peptides may beused to immunise animals to produce polyclonal or monoclonal antibodieswith specificity for peptides and/or proteins including the amino acidsequences. These antibodies may be then used to screen cDNA expressionlibraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group.Variations in genotype are important in commercial breeding programs, indetermining parentage, in diagnostics and fingerprinting, and the like.Genotypes can be readily described in terms of genetic markers. Agenetic marker identifies a specific region or locus in the genome. Themore genetic markers, the finer defined is the genotype. A geneticmarker becomes particularly useful when it is allelic between organismsbecause it then may serve to unambiguously identify an individual.Furthermore, a genetic marker becomes particularly useful when it isbased on nucleic acid sequence information that can unambiguouslyestablish a genotype of an individual and when the function encoded bysuch nucleic acid is known and is associated with a specific trait. Suchnucleic acids and/or nucleotide sequence information including singlenucleotide polymorphisms (SNPs), variations in single nucleotidesbetween allelic forms of such nucleotide sequence, may be used asperfect markers or candidate genes for the given trait.

Applicants have identified a number of SNPs of the nucleic acids ornucleic acid fragments of the present invention. These are indicated(marked with grey on the black background) in the figures that showmultiple alignments of nucleotide sequences of nucleic acid fragmentscontributing to consensus contig sequences. See for example, FIGS. 3, 6,9, 13, 16, 30, 37, 57, 60, 63, 79, 89, 92 and 104 hereto.

Accordingly, in a further aspect of the present invention, there isprovided a substantially purified or isolated nucleic acid or nucleicacid fragment including a single nucleotide polymorphism (SNP) from anucleic acid or nucleic acid fragment according to the presentinvention, for example a SNP from a nucleic acid sequence shown in FIGS.3, 6, 9, 13, 16, 30, 37, 57, 60, 63, 66, 67, 72, 78, 88, 94, 101 and 104hereto; or complements or sequences antisense thereto, and functionallyactive fragments and variants thereof. The invention further provides asubstantially purified or isolated nucleic acid or nucleic acid fragmentincluding a single nucleotide polymorphism (SNP) isolated by the methodof this invention.

In a still further aspect of the present invention there is provided amethod of isolating a nucleic acid or nucleic acid fragment of thepresent invention including a SNP, said method including sequencingnucleic acid fragments from a nucleic acid library. The method includesthe step of identifying the SNP.

The nucleic acid library may be of any suitable type and is preferably acDNA library.

The nucleic acid or nucleic acid fragment may be isolated from arecombinant plasmid or may be amplified, for example using polymerasechain reaction.

The sequencing may be performed by techniques known to those skilled inthe art.

In a still further aspect of the present invention, there is provideduse of the nucleic acids or nucleic acid fragments of the presentinvention including SNPs, and/or nucleotide sequence informationthereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided useof a nucleic acid or nucleic acid fragment of the present invention,and/or nucleotide sequence information thereof, as a molecular geneticmarker.

More particularly, nucleic acids or nucleic acid fragments according tothe present invention and/or nucleotide sequence information thereof maybe used as a molecular genetic marker for quantitative trait loci (QTL)tagging, QTL mapping, DNA fingerprinting and in marker assistedselection, particularly in clovers, alfalfa, ryegrasses and fescues.Even more particularly, nucleic acids or nucleic acid fragmentsaccording to the present invention and/or nucleotide sequenceinformation thereof may be used as molecular genetic markers in plantimprovement in relation to plant tolerance to abiotic stresses suchaluminium toxic acid soils; in relation to nutrient acquisitionefficiency including phosphorus; in relation to nitrogen fixation; inrelation to nodulation. Even more particularly, sequence informationrevealing SNPs in allelic variants of the nucleic acids or nucleic acidfragments of the present invention and/or nucleotide sequenceinformation thereof may be used as molecular genetic markers for QTLtagging and mapping and in marker assisted selection, particularly inclovers, alfalfa, ryegrasses and fescues.

In a still further aspect of the present invention there is provided aconstruct or vector including a nucleic acid or nucleic acid fragmentaccording to the present invention.

In a particularly preferred embodiment the construct or vector mayinclude nucleic acids or nucleic acid fragments encoding both CS orCS-like and MDH or MDH-like polypeptides.

In yet another preferred embodiment the construct or vector may includenucleic acids or nucleic acid fragments encoding both MDH or MDH-likeand PEPC or PEPC-like polypeptides.

In yet another preferred embodiment the construct or vector may includeboth CS or CS-like and PEPC or PEPC-like polypeptides.

In another preferred embodiment the construct or vector may includenucleic acids or nucleic acid fragments encoding all three of CS orCS-like, MDH or MDH-like and PEPC or PEPC-like polypeptides.

In a preferred embodiment of this aspect of the invention, the vectormay include one or more regulatory element such as a promoter, one ormore nucleic acids or nucleic acid fragments according to the presentinvention and one or more terminators; said one or more regulatoryelements, one or more nucleic acids or nucleic acid fragments and one ormore terminators being operatively linked.

In a preferred embodiment of the present invention the vector maycontain nucleic acids or nucleic acid fragments encoding both CS orCS-like and MDH or MDH-like polypeptides, operatively linked to aregulatory element or regulatory elements, such that both CS or CS-likeand MDH or MDH-like polypeptides are expressed.

In another preferred embodiment of the present invention the vector maycontain nucleic acids or nucleic acid fragments encoding both CS orCS-like and PEPC or PEPC-like polypeptides, operatively linked to aregulatory element or regulatory elements, such that both CS or CS-likeand PEPC or PEPC-like polypeptides are expressed.

In yet another particularly preferred embodiment of the presentinvention the vector or construct may contain nucleic acids or nucleicacid fragments encoding both MDH or MDH-like and PEPC or PEPC-likepolypeptides, operatively linked to a regulatory element or regulatoryelements, such that both MDH or MDH-like and PEPC or PEPC-likepolypeptides are expressed.

In another particularly preferred embodiment of the present inventionthe vector may contain nucleic acids or nucleic acid fragments encodingall three of CS or CS-like, MDH or MDH-like and PEPC or PEPC-like,operatively linked to a regulatory element or regulatory elements, suchthat all three of CS or CS-like, MDH or MDH-like and PEPC or PEPC-likepolypeptides are expressed.

The vector may be of any suitable type and may be viral or non-viral.The vector may be an expression vector. Such vectors includechromosomal, non-chromosomal and synthetic nucleic acid sequences, eg.derivatives of plant viruses; bacterial plasmids; derivatives of the Tiplasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmidfrom Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes;bacterial artificial chromosomes; binary bacterial artificialchromosomes; vectors derived from combinations of plasmids and phageDNA. However, any other vector may be used as long as it is replicable,integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type andmay be endogenous to the target plant cell or may be exogenous, providedthat they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoterswhich may be employed in the vectors of the present invention are wellknown to those skilled in the art. Factors influencing the choice ofpromoter include the desired tissue specificity of the vector, andwhether constitutive or inducible expression is desired and the natureof the plant cell to be transformed (eg. monocotyledon or dicotyledon).Particularly suitable constitutive promoters include the CauliflowerMosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, andthe rice Actin promoter. Particularly suitable tissue-specific promotersinclude root-prevalent promoters.

A variety of terminators which may be employed in the vectors of thepresent invention are also well known to those skilled in the art. Theterminator may be from the same gene as the promoter sequence or adifferent gene. Particularly suitable terminators are polyadenylationsignals, such as the CaMV 35S polyA and other terminators from thenopaline synthase (nos) and the octopine synthase (ocs) genes.

The vector, in addition to the regulatory element, the nucleic acid ornucleic acid fragment of the present invention and the terminator, mayinclude further elements necessary for expression of the nucleic acid ornucleic acid fragment, in different combinations, for example vectorbackbone, origin of replication (ori), multiple cloning sites, spacersequences, enhancers, introns (such as the maize Ubiquitin Ubi intron),antibiotic resistance genes and other selectable marker genes [such asthe neomycin phosphotransferase (npt2) gene, the hygromycinphosphotransferase (hph) gene, the phosphinothricin acetyltransferase(bar or pat) gene, the phospho-mannose isomerase (pmi) gene], andreporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. Thevector may also contain a ribosome binding site for translationinitiation. The vector may also include appropriate sequences foramplifying expression.

As an alternative to use of a selectable marker gene to provide aphenotypic trait for selection of transformed host cells, the presenceof the vector in transformed cells may be determined by other techniqueswell known in the art, such as PCR (polymerase chain reaction), Southernblot hybridisation analysis, histochemical GUS assays, northern andWestern blot hybridisation analyses.

Those skilled in the art will appreciate that the various components ofthe vector are operatively linked, so as to result in expression of saidnucleic acid or nucleic acid fragment. Techniques for operativelylinking the components of the vector of the present invention are wellknown to those skilled in the art. Such techniques include the use oflinkers, such as synthetic linkers, for example including one or morerestriction enzyme sites.

The vectors of the present invention may be incorporated into a varietyof plants, including monocotyledons (such as grasses from the generaLolium, Festuca, Paspalum, Pennisetum, Panicum and other forage andturfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (suchas Arabidopsis, tobacco, clovers, medics, eucalyptus, potato, sugarbeet,canola, soybean, chickpea) and gymnosperms. In a preferred embodiment,the vectors may be used to transform monocotyledons, preferably grassspecies such as ryegrasses (Lolium species) and fescues (Festucaspecies), more preferably perennial ryegrass, including forage- andturf-type cultivars. In an alternate preferred embodiment, the vectorsmay be used to transform dicotyledons, preferably forage legume speciessuch as clovers (Trifolium species) and medics (Medicago species), morepreferably white clover (Trifolium repens), red clover (Trifoliumpratense), subterranean clover (Trifoliuni subterraneum) and alfalfa(Medicago sativa). Clovers, alfalfa and medics are key pasture legumesin temperate climates throughout the world.

Techniques for incorporating the vectors of the present invention intoplant cells (for example by transduction, transfection ortransformation) are known to those skilled in the art. Such techniquesinclude Agrobacterium mediated introduction, electroporation to tissues,cells and protoplasts, protoplast fusion, injection into reproductiveorgans, injection into immature embryos and high velocity projectileintroduction to cells, tissues, calli, immature and mature embryos. Thechoice of technique will depend largely on the type of plant to betransformed.

Cells incorporating the vectors of the present invention may beselected, as described above, and then cultured in an appropriate mediumto regenerate transformed plants, using techniques well known in theart. The culture conditions, such as temperature, pH and the like, willbe apparent to the person skilled in the art. The resulting plants maybe reproduced, either sexually or asexually, using methods well known inthe art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plantcell, plant, plant seed or other plant part, including, e.g. transformedwith, a vector, nucleic acid or nucleic acid fragment of the presentinvention.

The plant cell, plant, plant seed or other plant part may be from anysuitable species, including monocotyledons, dicotyledons andgymnosperms. In a preferred embodiment the plant cell, plant, plant seedor other plant part may be from a monocotyledon, preferably a grassspecies, more preferably a ryegrass (Lolium species) or fescue (Festucaspecies), more preferably perennial ryegrass, including both forage- andturf-type cultivars. In an alternate preferred embodiment the plantcell, plant, plant seed or other plant part may be from a dicotyledon,preferably forage legume species such as clovers (Trifolium species) andmedics (Medicago species), more preferably white clover (Trifoliumrepens), red clover (Trifolium pratense), subterranean clover (Trifoliunsubterraneum) and alfalfa (Medicago sativa).

The present invention also provides a plant, plant seed or other plantpart, or a plant extract derived from a plant cell of the presentinvention.

The present invention also provides a plant, plant seed or other plantpart, or a plant extract derived from a plant of the present invention.

In a further aspect of the present invention there is provided a methodof modifying organic acid biosynthesis; of modifying organic acidsecretion; of modifying phosphorous and other nutrients acquisitionefficiency in plants; of modifying aluminium and acid soil tolerance inplants; of modifying nitrogen fixation and nodule function, said methodincluding introducing into said plant an effective amount of a nucleicacid or nucleic acid fragment according to the present invention.Preferably the nucleic acid or nucleic acid fragment is part of avector.

Using the methods and products of the present invention, organic acidbiosynthesis; organic acid secretion; phosphorous and other plantnutrient acquisition efficiency; aluminium and acid soil tolerance;nitrogen fixation and nodule function, may be increased or otherwisealtered, for example by incorporating additional copies of a sensenucleic acid or nucleic acid fragment of the present invention. They maybe decreased or otherwise altered, for example by incorporating anantisense nucleic acid or nucleic acid fragment of the presentinvention.

In a particularly preferred embodiment the method may includeintroducing into said plant nucleic acids or nucleic acid fragmentsencoding both CS or CS-like and MDH or MDH-like polypeptides.

In another preferred embodiment the method may include introducing intosaid plant nucleic acids or nucleic acid fragments encoding both CS orCS-like and PEPC or PEPC polypeptides.

In yet another preferred embodiment the method may include introducinginto said plant nucleic acids or nucleic acid fragments encoding bothMDH or MDH-like and PEPC or PEPC-like polypeptides.

In an even more preferred embodiment the method may include introducinginto said plant nucleic acids or nucleic acid fragments encoding allthree of CS or CS-like, MDH or MDH-like and PEPC or PEPC-likepolypeptides.

The present invention will now be more fully described with reference tothe accompanying Examples and drawings. It should be understood,however, that the description following is illustrative only and shouldnot be taken in any way as a restriction on the generality of theinvention described above.

In the Figures and Sequences

SEQ ID NO. 1 shows the consensus contig nucleotide sequence of LpCSa.

SEQ ID NO. 2 shows the deduced amino acid sequence of LpCSa.

FIG. 1 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpCSa.

SEQ ID NO. 11 shows the consensus contig nucleotide sequence of LpCSb.

SEQ ID NO. 12 shows the deduced amino acid sequence of LpCSb.

FIG. 2 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpCSb.

SEQ ID NO. 17 shows the nucleotide sequence of LpCSc.

SEQ ID NO. 18 shows the deduced amino acid sequence of LpCSc.

SEQ ID NO. 19 shows the nucleotide sequence of LpCSd.

SEQ ID NO. 20 shows the deduced amino acid sequence of LpCSd.

SEQ ID NO. 21 shows the consensus contig nucleotide sequence of LpMDHa.

SEQ ID NO. 22 shows the deduced amino acid sequence of LpMDHa.

FIG. 3 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpMDHa.

SEQ ID NO. 30 shows the consensus contig nucleotide sequence of LpMDHb.

SEQ ID NO. 31 shows the deduced amino acid sequence of LpMDHb.

FIG. 4 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpMDHb.

SEQ ID NO. 34 shows the nucleotide sequence of LpMDHc.

SEQ ID NO. 35 shows the deduced amino acid sequence of LpMDHc.

SEQ ID NO. 36 shows the nucleotide sequence of LpMDHd.

SEQ ID NO. 37 shows the deduced amino acid sequence of LpMDHd.

SEQ ID NO. 38 shows the nucleotide sequence of LpMDHe.

SEQ ID NO. 39 shows the deduced amino acid sequence of LpMDHe.

SEQ ID NO. 40 shows the consensus contig nucleotide sequence of LpMDHf.

SEQ ID NO. 41 shows the deduced amino acid sequence of LpMDHf.

FIG. 5 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpMDHf.

SEQ ID NO. 44 shows the nucleotide sequence of LpMDHg.

SEQ ID NO. 45 shows the deduced amino acid sequence of LpMDHg.

SEQ ID NO. 46 shows the consensus contig nucleotide sequence of LpMDHh.

SEQ ID NO. 47 shows the deduced amino acid sequence of LpMDHh.

FIG. 6 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpMDHh.

SEQ ID NO. 111 shows the nucleotide sequence of LpMDHi.

SEQ ID NO. 112 shows the deduced amino acid sequence of LpMDHi.

SEQ ID NO. 113 shows the nucleotide sequence of LpMDHj.

SEQ ID NO. 114 shows the deduced amino acid sequence of LpMDHj.

SEQ ID NO. 115 shows the consensus contig nucleotide sequence of LpMDHk.

SEQ ID NO. 116 shows the deduced amino acid sequence of LpMDHk.

FIG. 7 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpMDHk.

SEQ ID NO. 183 shows the nucleotide sequence of LpMDHl.

SEQ ID NO. 184 shows the deduced amino acid sequence of LpMDHl.

SEQ ID NO. 185 shows the nucleotide sequence of LpMDHm.

SEQ ID NO. 186 shows the deduced amino acid sequence of LpMDHm.

SEQ ID NO. 187 shows the nucleotide sequence of LpPEPCa.

SEQ ID NO. 188 shows the deduced amino acid sequence of LpPEPCa.

SEQ ID NO. 189 shows the consensus contig nucleotide sequence ofLpPEPCb.

SEQ ID NO. 190 shows the deduced amino acid sequence of LpPEPCb.

FIG. 8 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence LpPEPCb.

SEQ ID NO. 197 shows the nucleotide sequence of LpPEPCc.

SEQ ID NO. 198 shows the deduced amino acid sequence of LpPEPCc.

SEQ ID NO. 199 shows the nucleotide sequence of LpPEPCd.

SEQ ID NO. 200 shows the deduced amino acid sequence of LpPEPCd.

SEQ ID NO. 201 shows the nucleotide sequence of LpPEPCe.

SEQ ID NO. 202 shows the deduced amino acid sequence of LpPEPCe.

SEQ ID NO. 203 shows the nucleotide sequence of LpPEPCf.

SEQ ID NO. 204 shows the deduced amino acid sequence of LpPEPCf.

SEQ ID NO. 205 shows the consensus contig nucleotide sequence of TrMDHa.

SEQ ID NO. 206 shows the deduced amino acid sequence of TrMDHa.

FIG. 9 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHa.

SEQ ID NO. 218 shows the consensus contig nucleotide sequence of TrMDHb.

SEQ ID NO. 219 shows the deduced amino acid sequence of TrMDHb.

FIG. 10 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHb.

SEQ ID NO. 252 shows the consensus contig nucleotide sequence of TrMDHc.

SEQ ID NO. 253 shows the deduced amino acid sequence of TrMDHc.

FIG. 11 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHc.

SEQ ID NO. 271 shows the consensus contig nucleotide sequence of TrMDHd.

SEQ ID NO. 272 shows the deduced amino acid sequence of TrMDHd.

FIG. 12 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHd.

SEQ ID NO. 276 shows the consensus contig nucleotide sequence of TrMDHe.

SEQ ID NO. 277 shows the deduced amino acid sequence of TrMDHe.

FIG. 13 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHe.

SEQ ID NO. 288 shows the consensus contig nucleotide sequence of TrMDHf.

SEQ ID NO. 289 shows the deduced amino acid sequence of TrMDHf.

FIG. 14 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHf.

SEQ ID NO. 293 shows the consensus contig nucleotide sequence of TrMDHg.

SEQ ID NO. 294 shows the deduced amino acid sequence of TrMDHg.

FIG. 15 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHg.

SEQ ID NO. 297 shows the consensus contig nucleotide sequence of TrMDHh.

SEQ ID NO. 298 shows the deduced amino acid sequence of TrMDHh.

FIG. 16 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHh.

SEQ ID NO. 302 shows the consensus contig nucleotide sequence of TrMDHi.

SEQ ID NO. 303 shows the deduced amino acid sequence of TrMDHi.

FIG. 17 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrMDHi.

SEQ ID NO. 306 shows the nucleotide sequence of TrMDHj.

SEQ ID NO. 307 shows the deduced amino acid sequence of TrMDHj.

SEQ ID NO. 308 shows the nucleotide sequence of TrMDHk.

SEQ ID NO. 309 shows the deduced amino acid sequence of TrMDHk.

SEQ ID NO. 310 shows the consensus contig nucleotide sequence ofTrPEPCa.

SEQ ID NO. 311 shows the deduced amino acid sequence of TrPEPCa.

FIG. 18 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrPEPCa.

SEQ ID NO. 315 shows the consensus contig nucleotide sequence ofTrPEPCb.

SEQ ID NO. 316 shows the deduced amino acid sequence of TrPEPCb.

FIG. 19 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrPEPCb.

SEQ ID NO. 319 shows the consensus contig nucleotide sequence ofTrPEPCc.

SEQ ID NO. 320 shows the deduced amino acid sequence of TrPEPCc.

FIG. 20 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrPEPCc.

SEQ ID NO. 323 shows the nucleotide sequence of TrPEPCd.

SEQ ID NO. 324 shows the deduced amino acid sequence of TrPEPCd.

SEQ ID NO. 325 shows the nucleotide sequence of TrPEPCe.

SEQ ID NO. 326 shows the deduced amino acid sequence of TrPEPCe.

SEQ ID NO. 327 shows the consensus contig nucleotide sequence of TrCSa.

SEQ ID NO. 328 shows the deduced amino acid sequence of TrCSa.

FIG. 21 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrCSa.

SEQ ID NO. 336 shows the consensus contig nucleotide sequence of TrCSb.

SEQ ID NO. 337 shows the deduced amino acid sequence of TrCSb.

FIG. 22 shows the nucleotide sequences of the nucleic acid fragmentscontributing to the consensus contig sequence TrCSb.

FIG. 23 shows the plasmid map in pGEM-T Easy of TrMDH.

SEQ ID NO. 345 shows the nucleotide sequence of TrMDH.

SEQ ID NO. 346 shows the deduced amino acid sequence of TrMDH.

FIG. 24 shows the plasmid map of sense construct of TrMDH in the binaryvector pPZP221:35S².

FIG. 25 shows the plasmid map in pGEM-T Easy of TrPEPC.

SEQ ID NO. 347 shows the nucleotide sequence of TrPEPC.

SEQ ID NO. 348 shows the deduced amino acid sequence of TrPEPC.

FIG. 26 shows the plasmid map of sense construct of TrPEPC in the binaryvector pPZP221:35S².

FIG. 27 shows the plasmid map in pGEM-T Easy of TrCSa.

SEQ ID NO. 349 shows the nucleotide sequence of TrCSa.

SEQ ID NO. 350 shows the deduced amino acid sequence of TrCSa.

FIG. 28 shows the plasmid map of sense construct of TrCSa in the binaryvector pPZP221:35S².

FIG. 29 shows the plasmid map in pGEM-T Easy of TrCSb.

SEQ ID NO. 351 shows the nucleotide sequence of TrCSb.

SEQ ID NO. 352 shows the deduced amino acid sequence of TrCSb.

FIG. 30 shows the plasmid map of sense construct of TrCSb in the binaryvector pPZP221:35S².

FIG. 31 shows the plasmid map in pGEM-T Easy of TrCSd.

SEQ ID NO. 353 shows the nucleotide sequence of TrCSd.

SEQ ID NO. 354 shows the deduced amino acid sequence of TrCSd.

FIG. 32 shows the plasmid map of sense construct of TrCSd in the binaryvector pPZP221:35S².

FIG. 33 shows the plasmid maps of the modular vector system comprising abinary base vector and 7 auxiliary vectors.

FIG. 34 shows an example of the modular binary transformation vectorsystem comprising plasmid maps of the binary transformation vectorbackbone and 4 expression cassettes for combinatorial expression ofchimeric CS and MDH and PEPC genes in auxiliary vectors (A) and theplasmid map of the T-DNA region of the final binary transformationvector (B).

FIG. 35 shows the results of RT-PCR experiments performed as describedin Example 6. Samples were isolated from: L, leaf; S, stolon; St, stolontip; R, root; Rt, root tip. −C: negative (no reverse transcriptase)control; +C, positive (plasmid) control. The numbers indicate cyclenumbers. A: phosphate transporter homolog; B: root iron transporterhomolog.

FIG. 36 shows the screening of a white clover BAC library using thephosphate transporter cDNA as a probe (A); Southern hybridisation blotof six BAC clones identified in A using the same probe (B); physical mapof the phosphate transporter genomic region including the coding regionand the promoter region (C).

FIG. 37 shows white clover cotyledons, various stages of selection ofplantlets transformed with a binary transformation vector constructed asdescribed in Examples 4 and 5, transgenic white clover on root-inducingmedium, and white clover plants transformed with genes involved inorganic acid biosynthesis.

FIG. 38 shows the molecular analysis of transgenic white clover plantsfor the presence of the chimeric MDH gene with real time PCRamplification plot and agarose gel of PCR product.

FIG. 39 shows the molecular analysis of transgenic white clover plantsfor the presence of the chimeric PEPC gene with real time PCRamplification plot and agarose gel of PCR product.

FIG. 40 shows the molecular analysis of transgenic white clover plantsfor the presence of the chimeric CS gene with real time PCRamplification plot and agarose gel of PCR product.

EXAMPLE 1

Preparation of cDNA Libraries, Isolation and Sequencing of cDNAs Codingfor CS, CS-like, MDH, MDH-like, PEPC and PEPC-like Polypeptides fromWhite Clover (Trifolium repens) and Perennial Ryegrass (Lolium perenne)

cDNA libraries representing mRNAs from various organs and tissues ofwhite clover (Trifolium repens) and perennial ryegrass (Lolium perenne)were prepared. The characteristics of the white clover and perennialryegrass libraries, respectively, are described below (Tables 1 and 2).TABLE 1 cDNA libraries from white clover (Trifolium repens) LibraryOrgan/Tissue 01wc Whole seedling, light grown 02wc Nodulated root 3, 5,10, 14, 21 & 28 day old seedling 03wc Nodules pinched off roots of 42day old rhizobium inoculated white clover 04wc Nodulated white clovercut leaf and stem collected after 0, 1, 4, 6 & 14 h after cutting 05wcNon-nodulated Inflorescences: <50% open, not fully open and fully open06wc Dark grown etiolated 07wc Inflorescence - very early stages, stemelongation, <15 petals, 15-20 petals 08wc seed frozen at −80° C.,imbibed in dark overnight at 10° C. 09wc Drought stressed plants 10wcAMV infected leaf 11wc WCMV infected leaf 12wc Phosphorus starved plants13wc Vegetative stolon tip 14wc stolon root initials 15wc Senescingstolon 16wc Senescing leaf

TABLE 2 cDNA libraries from perennial ryegrass (Lolium perenne) LibraryOrgan/Tissue 01rg Roots from 3-4 day old light-grown seedlings 02rgLeaves from 3-4 day old light-grown seedlings 03rg Etiolated 3-4 day olddark-grown seedlings 04rg Whole etiolated seedlings (1-5 day old and 17days old) 05rg Senescing leaves from mature plants 06rg Whole etiolatedseedlings (1-5 day old and 17 days old) 07rg Roots from mature plantsgrown in hydroponic culture 08rg Senescent leaf tissue 09rg Wholetillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after harvesting)10rg Embryogenic suspension-cultured cells 11rg Non-embryogenicsuspension-cultured cells 12rg Whole tillers and sliced leaves (0, 1, 3,6, 12 and 24 h after harvesting) 13rg Shoot apices including vegetativeapical meristems 14rg Immature inflorescences including different stagesof inflorescence meristem and inflorescence development 15rg Defattedpollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2Asubtracted) 17rg Senescing leaves and tillers 18rg Drought-stressedtillers (pseudostems from plants subjected to PEG-simulated droughtstress) 19rg Non-embryogenic suspension-cultured cells subjected toosmotic stress (grown in media with half-strength salts) (1, 2, 3, 4, 5,6, 24 and 48 h after transfer) 20rg Non-embryogenic suspension-culturedcells subjected to osmotic stress (grown in media with double-strengthsalts) (1, 2, 3, 4, 5, 6, 24 and 48 h after transfer) 21rgDrought-stressed tillers (pseudostems from plants subjected toPEG-simulated drought stress) 22rg Spikelets with open and maturingflorets 23rg Mature roots (specific subtraction with leaf tissue)

The cDNA libraries may be prepared by any of many methods available. Forexample, total RNA may be isolated using the Trizol method (Gibco-BRL,USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following themanufacturers' instructions. cDNAs may be generated using the SMART PCRcDNA synthesis kit (Clontech, USA), cDNAs may be amplified by longdistance polymerase chain reaction using the Advantage 2 PCR Enzymesystem (Clontech, USA), cDNAs may be cleaned using the GeneClean spincolumn (Bio 101, USA), tailed and size fractionated, according to theprotocol provided by Clontech. The cDNAs may be introduced into thepGEM-T Easy Vector system 1 (Promega, USA) according to the protocolprovided by Promega. The cDNAs in the pGEM-T Easy plasmid vector aretransfected into Escherichia coli Epicurian coli XL10-Gold ultracompetent cells (Stratagene, USA) according to the protocol provided byStratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors forfirst preparing the cDNA libraries in Uni-ZAP XR vectors according tothe manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif., USA). The Uni-ZAP XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts will be contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase(New England Biolabs), followed by transfection into E. coli DH10B cellsaccording to the manufacturer's protocol (GIBCO BRL Products).

Once the cDNA inserts are in plasmid vectors, plasmid DNAs are preparedfrom randomly picked bacterial colonies containing recombinant plasmids,or the insert cDNA sequences are amplified via polymerase chain reactionusing primers specific for vector sequences flanking the inserted cDNAsequences. Plasmid DNA preparation may be performed robotically usingthe Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocolprovided by Qiagen. Amplified insert DNAs are sequenced indye-terminator sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”). The resulting ESTs are analysedusing an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2

DNA Sequence Analyses

The cDNA clones encoding CS, CS-like, MDH, MDH-like, PEPC and PEPC-likepolypeptides were identified by conducting BLAST (Basic Local AlignmentSearch Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches.The cDNA sequences obtained were analysed for similarity to all publiclyavailable DNA sequences contained in the eBioinformatics nucleotidedatabase using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the SWISS-PROT protein sequence databaseusing BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics3:266-272) provided by the NCBI.

The cDNA sequences obtained and identified were then used to identifyadditional identical and/or overlapping cDNA sequences generated usingthe BLASTN algorithm. The identical and/or overlapping sequences weresubjected to a multiple alignment using the CLUSTALw algorithm, and togenerate a consensus contig sequence derived from this multiple sequencealignment. The consensus contig sequence was then used as a query for asearch against the SWISS-PROT protein sequence database using the BLASTxalgorithm to confirm the initial identification.

EXAMPLE 3

Identification and Full-length Sequencing of cDNAs Encoding CS, MDH andPEPC Polypeptides

To fully characterise for the purposes of the generation of probes forhybridisation experiments and the generation of transformation vectors,a set of cDNAs encoding white clover CS, MDH and PEPC polypeptides wasidentified and fully sequenced.

Full-length cDNAs were identified from our EST sequence database usingrelevant published sequences (NCBI databank) as queries for BLASTsearches. Full-length cDNAs were identified by alignment of the queryand hit sequences using Sequencher (Gene Codes Corp., Ann Arbor, Mich.48108, USA). The original plasmid was then used to transform chemicallycompetent XL-1 cells (prepared in-house, CaCl₂ protocol). After colonyPCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive coloniesper transformation were picked for initial sequencing with M13F and M13Rprimers. The resulting sequences were aligned with the original ESTsequence using Sequencher to confirm identity and one of the threeclones was picked for full-length sequencing, usually the one with thebest initial sequencing result.

Sequencing of all cDNAs was completed by primer walking, i.e.oligonucleotide primers were designed to the initial sequence obtainedusing M13F and M13R oligonucleotide primers and used for furthersequencing. The sequences of the oligonucleotide primers are shown inTable 2.

Contigs were then assembled in Sequencher. The contigs include thesequences of the SMART primers used to generate the initial cDNA libraryas well as pGEM-T Easy vector sequence up to the EcoRI cut site both atthe 5′ and 3′ end.

Plasmid maps and the full cDNA sequences of TrCSa, TrCSb, TrCSd, TrMDHand TrPEPC polypeptides were obtained (SEQ ID NOS: 1, 2, 12, FIG. 2, SEQID NOS: 19, 20 20, FIG. 3, SEQ ID NOS: 30, 34, 35, 38, 39, FIG. 5, SEQID NOS: 44, 47 and FIG. 6). TABLE 3 List of primers used for sequencingof the full-length cDNAs encoding CS, MDH and PEPC Seq. gene sequencingID name clone ID primer NO: primer sequence (5′>3′) TrCSa 05wc1HsB0805wc1HsB08.f1 355 TTGCCCGAGGCTATACTGTGGC 05wc1HsB08.f2 356CAGCTCACCTAGTTGCTAG 05wc1HsB08.f3 357 CCATGGCCTAATGTTGATGC 05wc1HsB08.r1358 TTGGCCTTTCAAGTGGCATTCC 05wc1HsB08.r2 359 CAGAATGGGAGGCACGACTTC05wc1HsB08.r3 360 ATGTGAGCATAGTTTGCACC TrCSb 05wc2HsD09 05wc2HsD09.f1361 GACTGCCAGAAAACACTTCCAGG 05wc2HsD09.f2 362 ATGACTGCTTTAGTGTGG05wc2HsD09.r1 363 CTCAAGTTTCTCCAGTGTGACAC 05wc2HsD09.r2 364TGACTTATGTATCCCACC 05wc2HsD09.r3 365 GCTCTGAATGGTTTAGCTGG TrCSd10wc1BsF10 10wc1BsF10.f1 366 GCACTGCCTGTTTCTGCTCATCC 10wc1BsF10.f2 367AGCCAACTTATGAGGATAGC 10wc1BsF10.r1 368 CTCCAATACTCCTCGCGACGCC10wc1BsF10.r2 369 AGGCACAACCTGGCCACTG 10wc1BsF10.r3 370ACGTTGCCACCTTCATGATC TrMDH 13wc1NsD01 13wc1NsD01.f1 371GTTGTTATACCTGCTGGTGTT 13wc1NsD01.r1 372 CTCACTCAACCCTTGGAGAT TrPEPC15wc1DsH12 15wc1DsH12.f1 373 TCCTAAGAAACTTGAAGAGCTCGG 15wc1DsH12.f2 374AGATGTTTGCTTACTAGC 15wc1DsH12.r1 375 GCCAGCAGCAATACCCTTCATGG15wc1DsH12.r2 376 TTGCTTCTCAACTGTTCC

EXAMPLE 4

Development of Binary Transformation Vectors Containing Chimeric Geneswith cDNA Sequences Encoding CS, MDH and PEPC

To alter the expression of the polypeptides involved in organic acidbiosynthesis to improve phosphorus acquisition efficiency as well asaluminium and acid soil tolerance in forage plants, a set of sensebinary transformation vectors was produced.

The pPZP221 binary transformation vector (Hajdukiewicz et al., 1994) wasmodified to contain the 35S² cassette from pKYLX71:35S² (Schardl et al.,1987) as follows: pKYLX71:35S² was cut with Clal. The 5′ overhang wasfilled in using Klenow and the blunt end was A-tailed with Taqpolymerase. After cutting with EcoRI, the 2 kb fragment with anEcoRI-compatible and a 3′-A tail was gel-purified. pPZP221 was cut withHindll and the resulting 5′ overhang filled in and T-tailed with Taqpolymerase. The remainder of the original pPZP221 multi-cloning site wasremoved by digestion with EcoRI, and the expression cassette cloned intothe EcoRI site and the 3′ T overhang restoring the HindlIl site. Thisbinary vector contains between the left and right border the plantselectable marker gene aacC1 under the control of the 35S promoter and35S terminator and the pKYLX71:35S²-derived expression cassette with aCaMV 35S promoter with a duplicated enhancer region and an rbcSterminator.

A GATEWAY® cloning cassette (Invitrogen) was introduced into themulticloning site of the pPZP221:35S² vector obtained as describedfollowing the manufacturer's protocol.

cDNA fragments were generated by high fidelity PCR with a proofreadingDNA polymerase using the original pGEM-T Easy plasmid cDNA as atemplate. The primers used (Table 3) contained attB sequences for usewith recombinases utilising the GATEWAY® system (Invitrogen). Theresulting PCR fragments were used in a recombination reaction withpDONR® vector (Invitrogen) to generate entry vectors. In a furtherrecombination reaction, the cDNAs encoding the open reading framesequences were transferred from the entry vector to the GATEWAY®-enabledpPZP221:35S² vector.

The orientation of the constructs (sense or antisense) was checked byrestriction enzyme digest and sequencing which also confirmed thecorrectness of the sequence. Transformation vectors containing chimericgenes using full-length open reading frame cDNAs encoding white cloverTrCSa, TrCSb, TrCSd, TrMDH and TrPEPC proteins in sense orientationunder the control of the CaMV 35S² promoter were generated (SEQ ID NOS:11, 18, 22, FIG. 4, SEQ ID NOS: 37, 41 and 46). TABLE 4 List of primersused to PCR-amplify the open reading frames of cDNAs encoding CS, MDHand PEPC gene Seq ID name clone ID primer No primer sequence (5′>3′)TrCSa 05wc1HsB08 05wc1HsB08f 377 GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATCTTAATGGCGTTCT TTCG 05wc1HsB08r 378 GGGGACCACTTTGTACAAGAAAGCTGGGTTTTCAATTTTAGGACGATG CG TrCSb 05wc2HsD09 05wc2HsD09f 379GGGGACAAGTTTGTACAAAAAAG CAGGCTTTGTTGATTGATCTTAAT GGC 05wc2HsD09r 380GGGGACCACTTTGTACAAGAAAGC TGGGTTAGTAATCCACAGATAACC G TrCSd 10wc1BsF1010wc1BsF10f 381 GGGGACAAGTTTGTACAAAAAAG CAGGCTCTAGATTGTTGATTGATCTAAATGGC 10wC1BsF10r 382 GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGATTCAATTTTAGGAT GATGCACC TrMDH 13wc1NsD1 13wc1NsD01f 383GGGGACAAGTTTGTACAAAAAAG CAGGCTCTAGAAATTCCCATTACC ATTCATTCC 13wc1NsD01r384 GGGGACCACTTTGTACAAGAAAGC TGGGTCTAGATTGACATTCTCTCG CATGGACGC TrPEPC15wc1DsH2 15wc1DsH12f 385 GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAGAAGGAGTGAATTGC TCC 15wc1DsH12r 386 GGGGACCACTTTGTACAAGAAAGCTGGGTATGATATCTTAGCACACAC TTAAC

EXAMPLE 5

Development of Binary Transformation Vectors Containing Chimeric Geneswith a Combination of 2 or More cDNA Sequences Encoding CS, MDH and PEPC

To alter the expression of the polypeptides involved in organic acidbiosynthesis to improve phosphorus acquisition efficiency as well asaluminium and acid soil tolerance in forage plants, a modular binarytransformation vector system was used (FIG. 33). The modular binaryvector system enables simultaneous integration of up to seven transgenesthe expression of which is controlled by individual promoter andterminator sequences into the plant genome (Goderis et al., 2002).

The modular binary vector system consists of a pPZP200-derived vector(Hajdukiewicz et al., 1994) backbone containing within the T-DNA anumber of simultaneous integration of up to seven transgenes theexpression of which is controlled by individual promoter and terminatorsequences into the plant genome. (Goderis et al., 2002).

The modular binary vector system consists of a pPZP200-derived vector(Hajdukiewicz et al., 1994) backbone containing within the T-DNA anumber of unique restriction sites recognised by homing endonucleases.The same restriction sites are present in pUC18-based auxiliary vectorsflanking standard multicloning sites. Expression cassettes comprising aselectable marker gene sequence or a cDNA sequence to be introduced intothe plant under the control of regulatory sequences like promoter andterminator can be constructed in the auxiliary vectors and thentransferred to the binary vector backbone utilising the homingendonuclease restriction sites. Up to seven expression cassettes canthus be integrated into a single binary transformation vector. Thesystem is highly flexible and allows for any combination of cDNAsequence to be introduced into the plant with any regulatory sequence.

For example, a selectable marker cassette comprising the nos promoterand nos terminator regulatory sequences controlling the expression ofthe nptII gene was PCR-amplified using a proofreading DNA polymerasefrom the binary vector pKYLX71:35S² and directionally cloned into theAgel and NotI sites of the auxiliary vector pAUX3166. Equally, otherselectable marker cassettes can be introduced into any of the auxiliaryvectors.

In another example, the expression cassette from the binary vector pWM5consisting of the ASSU promoter and the tob terminator was PCR-amplifiedwith a proofreading DNA polymerase and directionally cloned into theAgeI and NotI sites of the auxiliary vector pAUX3169. Equally, otherexpression cassettes can be introduced into any of the auxiliaryvectors.

In yet another example, the expression cassette from the direct genetransfer vector pDH51 was cut using EcoRI and cloned directly into theEcoRI site of the auxiliary vector pAUX3132. TABLE 5 List of primersused to PCR-amplify plant selectable marker cassettes and the regulatoryelements used to control the expression of CS, MDH and PEPC genesexpression Seq. cassette primer ID No. primer sequence (5′>3′)nos::nptII-nos forward 387 ATAATAACCGGTTGATCATGAGCGGAGAATTA AGGG reverse388 ATAATAGCGGCCGCTAGTAACATAGATGACAC CGCG 35S::aacC1-35S forward 389AATAGCGGCCGCGATTTAGTACTGGATTTTGG reverse 390AATAACCGGTACCCACGAAGGAGCATCGTGG 35S²::rbcS forward 391ATAATAACCGGTGCCCGGGGATCTCCTTTGCC reverse 392ATAATAGCGGCCGCATGCATGTTGTCAATCAA TTGG assu::tob forward 393TAATACCGGTAAATTTATTATGRGTTTTTTTCC G reverse 394TAATGCGGCCGCTAAGGGCAGCCCATACAAAT GAAGC

The expression cassettes were further modified by introducing a GATEWAY®cloning cassette (Invitrogen) into the multicloning site of therespective pAUX vector following the manufacturer's protocol. In arecombination reaction, the cDNAs encoding the open reading framesequences were transferred from the entry vector obtained as describedin Example 4 to the GATEWAY®-enabled pAUX vector. Any combination of theregulatory elements with cDNA sequences of TrCSa, TrCSb, TrCSd, TrMDHand TrPEPC can be obtained. One typical example is given in FIG. 34 withexpression cassettes comprising the nptll plant selectable marker,TrPEPC, TrCSa and TrMDH.

Complete expression cassettes comprising any combination of regulatoryelements and cDNA sequences to be introduced into the plant were thencut from the auxiliary vectors using the respective homing endonucleaseand cloned into the respective restriction site on the binary vectorbackbone. After verification of the construct by nucleotide sequencing,the binary transformation vector comprising a number of expressioncassettes was used to generate transgenic white clover plants.

EXAMPLE 6

Isolation of Regulatory Elements to Direct Expression of Chimeric GenesEncoding CS, MDH and PEPC Involved in Organic Acid Biosynthesis

To direct the expression of chimeric white clover genes TrCSa, TrCSb,TrCSd, TrMDH and TrPEPC involved in organic acid biosynthesis tospecific tissues, regulatory elements showing specificity for expressionin root or root tip tissue were identified and isolated.

Using the BLASTn algorithm, white clover EST sequence collectionsprepared as detailed in Examples 1 and 2 were searched with nucleotidesequences representing genes with known root-specific expressionidentified in GenBank as queries. Suitable candidate ESTs wereidentified and oligonucleotide primers for reverse transcription-PCR(RT-PCR) were designed (see Table 4). TABLE 6 Oligonucleotide primersused in reverse transcription-PCR to confirm tissue specificity ofcandidate white clover ESTs gene forward primer (5′->3′) reverse primer(5′->3′) histone (internal control) CCGATTCCGTTTCAATG GCCATCCTTAACCCTAAGGCTCGTA CACGT SEQ ID No: 395 SEQ ID No: 396 white clover phosphateTTGCATTTGCTTGGAAC GCAAGAGCAAACATGAA transporter homolog AACTAG ACCA SEQID No: 397 SEQ ID No: 398 white clover root iron ATGGGTCTTGGTGGTTGGCAGCAAGAAGATCAAC transporter homolog CA CAAAGCCA SEQ ID No: 398 SEQ IDNo: 400

Total RNA for RT-PCR experiments was isolated from a leaf, stolon,stolon tip, root and root tip of white clover plants grown in theglasshouse using the TRIZOL method.

Reverse transcription was performed using SuperScriptII (Invitrogen)following the supplier's instructions. Preliminary PCR reactions usingDynazyme as the DNA polymerase were set up to determine the correctamount of template using the PCR primers for the internal control(histone). The results of this preliminary PCR were used to set upanother round of PCR to determine the optimum number of cycles forlinear amplification. The final PCR amplifications were performed usingthe following cycling conditions: 94° C., 4 min., 1 time; 94° C., 15sec., 60° C., 30 sec., 72° C., 2 min.,×times; 72° C., 10 min., 1 time.The number of cycles in the amplification (×) was found to be dependenton the relative abundance of transcript and had to be optimised for eachtemplate.

RT-PCR results using a white clover histone gene as an internalconstitutively expressed control confirmed the tissue-specificity of twocandidate ESTs to be root-prevalent (FIG. 35 A and B). These were aphosphate transporter homolog (clone name 02wc1DsG07) and a root irontransporter homolog (clone name 05wc1IsB 11).

A spotted white clover BAC library consisting of 50,304 clones with anestimated 99% genome coverage (6.3 genome equivalents) was screenedusing the phosphate transporter homolog EST nucleotide sequence as aprobe. A number of positive BAC clones could be identified (FIG. 36 A).After Southern hybridisation blotting (FIG. 36 B) a 7.5 kb EcoRV genomicDNA fragment was selected and fully sequenced. The fragment containedthe phosphate transporter homolog open reading frame and 4 kb ofupstream sequence including the promoter region. A physical map of thegenomic DNA fragment including the promoter region is shown in FIG. 36C.

EXAMPLE 7

Production by Agrobacterium-mediated Transformation and Analysis ofTransgenic White Clover Plants Carrying Chimeric Genes Encoding CS, MDHand PEPC Involved in Organic Acid Biosynthesis

A set of binary transformation vectors carrying chimeric white clovergenes to alter the expression of the polypeptides involved in organicacid biosynthesis to improve phosphorus acquisition efficiency as wellas aluminium and acid soil tolerance in forage plants were produced asdetailed in Examples 4 and 5.

Agrobacterium-mediated gene transfer experiments were performed usingthese transformation vectors.

The production of transgenic white clover plants carrying the whiteclover TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC cDNAs, either singly or incombination, is described here in detail (Table 7).

Preparation of Agrobacterium

Agrobacterium tumefaciens strain AGL-1 transformed with one of thebinary vector constructs detailed in Example 6 were streaked on LBmedium containing 50 μg/ml rifampicin and 50 μg/ml kanamycin and grownat 27° C. for 48 hours. A single colony was used to inoculate 5 ml of LBmedium containing 50 μg/ml rifampicin and 50 μg/ml kanamycin and grownover night at 27° C. and 250 rpm on an orbital shaker. The overnightculture was used as an inoculum for 500 ml of LB medium containing 50μg/ml kanamycin only. Incubation was over night at 27° C. and 250 rpm onan orbital shaker in a 21 Erlenmeyer flask.

Preparation of White Clover Seeds

1 spoon of seeds (ca. 500) was placed into a 280 μm mesh size sieve andwashed for 5 min under running tap water, taking care not to wash seedsout of sieve. In a laminar flow hood, seeds were transferred with thespoon into an autoclaved 100 ml plastic culture vessel. A magneticstirrer (wiped with 70% EtOH) and ca. 30 ml 70% EtOH were added, and theseeds were stirred for 5 min. The EtOH was discarded and replaced by 50ml 1.5% sodium hypochlorite. The seeds were stirred for an additional45-60 min on a magnetic stirrer. The sodium hypochlorite was thendiscarded and the seeds rinsed 3 to 4 times with autoclaved ddH₂O.Finally 30 ml of ddH₂O were added, and seeds incubated over night at10-15° C. in an incubator.

Agrobacterium-mediated Transformation of White Clover

The seed coat and endosperm layer of the white clover seeds prepared asabove were removed with a pair of 18 G or 21 G needles. The cotyledonswere cut from the hypocotyl leaving a ca. 1.5 mm piece of the cotyledonstalk. The cotyledons were transferred to a petridish containing ddH₂O.After finishing the isolation of clover cotyledons, ddH₂O in thepetridish was replaced with Agrobacterium suspension (diluted to anOD₆₀₀=0.2-0.4). The petridish was sealed with its lid and incubated for40 min at room temperature.

After the incubation period, each cotyledon was transferred to papertowel using the small dissection needle, dried and plated ontoregeneration medium RM73. The plates were incubated at 25° C. with a 16h light/8 h dark photoperiod. On day 4, the explants were transferred tofresh regeneration medium. Cotyledons transformed with Agrobacteriumwere transferred to RM73 containing cefotaxime (antibacterial agent) andgentamycin. The dishes were sealed with Parafilm and incubated at 25° C.under a 16/8 h photoperiod. Explants were subcultured every three weeksfor a total of nine weeks onto fresh RM 73 containing cefotaxime andgentamycin. Shoots with a green base were then transferred toroot-inducing medium RIM. Roots developed after 1-3 weeks, and plantletswere transferred to soil when the roots were well established.

Preparation of Genomic DNA for Real-time PCR and Analysis for thePresence of Transgenes

3-4 leaves of white clover plants regenerated on selective medium wereharvested and freeze-dried. The tissue was homogenised on a Retsch MM300mixer mill, then centrifuged for 10 min at 1700×g to collect celldebris. Genomic DNA was isolated from the supernatant using WizardMagnetic 96 DNA Plant System kits (Promega) on a Biomek FX (BeckmanCoulter). 5 μl of the sample (50 μl) were then analysed on an agarosegel to check the yield and the quality of the genomic DNA.

Genomic DNA was analysed for the presence of the transgene by real-timePCR using SYBR Green chemistry. PCR primer pairs were designed usingMacVector (Accelrys) or PrimerExpress (ABI). The forward primer waslocated within the 35S² promoter region and the reverse primer withinthe transgene to amplify products of approximately 150-250 bp asrecommended. The positioning of the forward primer within the 35S²promoter region guaranteed that endogenous genes in white clover werenot detected.

5 μl of each genomic DNA sample was run in a 50 μl PCR reactionincluding SYBR Green on an ABI 7700 (Applied Biosystems) together withsamples containing DNA isolated from wild type white clover plants(negative control), samples containing buffer instead of DNA (buffercontrol) and samples containing the plasmid used for transformation(positive plasmid control). Cycling conditions used were 2 min. at 50°C., 10 min. at 95° C., and then 40 cycles of 15 sec. at 95° C., 1 min.at 60° C.

Preparation of Genomic DNA and Analysis of DNA for Presence and CopyNumber of Transgene by Southern Hybridisation Blotting

Genomic DNA for Southern hybridisation blotting was obtained from leafmaterial of white clover plants following the CTAB method. Southernhybridisation blotting experiments were performed following standardprotocols as described in Sambrook et al. (1989). In brief, genomic DNAsamples were digested with appropriate restriction enzymes and theresulting fragments separated on an agarose gel. After transfer to amembrane, a cDNA fragment representing a transgene or selectable markergene was used to probe the size-fractionated DNA fragments.Hybridisation was performed with either radioactively labelled probes orusing the non-radioactive DIG labelling and hybridisation protocol(Boehringer) following the manufacturer's instructions.

Plants were obtained after transformation with all chimeric constructsand selection on medium containing gentamycin. Details of plant analysisare given in Table 5 and FIGS. 38, 39 and 40. TABLE 7 Transformation ofwhite clover with binary transformation vectors comprising cDNAsencoding CS, MDH and PEPC involved in organic acid biosyntheses,selection and molecular analysis of regenerated plants. cotyledonsselection into copy number construct transformed RIM soil QPCR-positiveSouthern range pPZP221-35S2::TrMDH 2739 72 45 43 n/d pPZP221-35S2::TrCS2550 39 7 nd n/d pPZP221-35S2::TrPEPC 2730 44 10 nd n/d

REFERENCES

-   Altschul, S. F., Gish, W., Miller, W., Myers, E. W.,    Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol.    Biol. 215, 403-410.-   Frohman, M. A., Dush, M. K., Martin, G. R. (1988) Rapid production    of full-length cDNAs from rare transcripts: amplification using a    single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci.    USA 85, 8998.-   Gish, W., States, D. J. (1993) Identification of protein coding    regions by database similarity search. Nature Genetics 3, 266-272.-   Goderis, I., De Bolle, M. F. C., Francois, I., Wouters, P. F. J.,    Broekaert, W. F., and Cammue, B. P. A. (2002) A set of modular plant    transformation vectors allowing flexible insertion of up to six    expression units. Plant Molecular Biology 50, 17-27.-   Hajdukiewicz P, Svab Z, Maliga P. (1994) The small, versatile pPZP    family of Agrobacterium binary vectors for plant transformation.    Plant Mol Biol. 25, 989-94.-   Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., Davis, M. M.    (1989). Polymerase chain reaction with single-sided specificity:    Analysis of T-cell receptor delta chain. Science 243, 217-220.-   Ohara, O., Dorit, R. L., Gilbert, W. (1989). One-sided polymerase    chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA    86, 5673-5677-   Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular    Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press-   Schardl, C. L., Byrd, A. D., Benzion, G., Altschuler, M. A.,    Hildebrand, D. F., Hunt, A. G. (1987) Design and construction of a    versatile system for the expression of foreign genes in plants. Gene    61, 1-11

Finally, it is to be understood that various alterations, modificationsand/or additions may be made without departing from the spirit of thepresent invention as outlined herein.

1-29. (canceled)
 30. A substantially purified or isolated nucleic acidor nucleic acid fragment encoding an organic acid biosynthesis enzymepolypeptide selected from the group consisting of a citrate synthase(CS) polypeptide; a CS-like polypeptide; a malate dehydrogenase (MDH)polypeptide; a MDH-like polypeptide; a phosphoenolpyruvate carboxylase(PEPC) polypeptide; and a PEPC-like polypeptide; or a functionallyactive fragment or variant of such a polypeptide, from a clover(Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca)species.
 31. A nucleic acid or nucleic acid fragment according to claim30 wherein said nucleic acid or nucleic acid fragment is from whiteclover (Trifolium repens) or perennial ryeglass (Lolium perenne).
 32. Anucleic acid or nucleic acid fragment encoding a CS or CS-likepolypeptide and including a nucleotide sequence selected from thegloupconsisting of (a) sequences shown in (SEQ ID NOS 1, 3 to 10, 11, 13to 16, 17, 19, 327, 329 to 335, 336, 338 to 344, 349, 351, and 353; (b)complements of the sequences recited in (a); (c) sequences antisense tothe sequences recited in (a) and (b); (d) functionally active fragmentsand variants of the sequences recited in (a), (b), and (c); and (e) RNAsequences corresponding to the sequences recited in (a), (b), (c) and(d).
 33. A nucleic acid or nucleic acid fragment encoding a MDH orMDH-like polypeptide and including a nucleotide sequence selected fromthe group consisting of (a) sequence shown in SEQ ID NOS 21, 23 to 29;30, 32 to33, 34, 36, 38, 40, 42to 43, 44, 46, 48 to 110, 111, 113, 115,117 to 182, 183, 185, 205, 207 to 217, 218, 220 to 251, 252, 254 to 270,271, 273 to 275, 276, 278 to 287, 288, 290 to 292, 293, 295 to 296, 297,299 to 301, 304 to 305, 306, and 308; (b) complements of the sequencesrecited in (a); (c) sequences antisense to the sequences recited in (a)and (b); (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c); and (e) RNA sequences corresponding to thesequences recited in (a), (b), (c) and (d).
 34. A nucleic acidornucleicacidfi-agment encoding a PEPC or PEPC-like polypeptide and including anucleotide sequence selected from the group consisting of (a) sequencesshown in SEQ ID NOS 187, 189, 191 to 197, 199, 201, 903, 310, 312 to314, 315, 317 to 318, 319, 321 to 399, 393, 325 and 347; (b) complementsof the sequences recited in (a); (c) sequences antisense to thesequences recited in (a) and (b); (d) functionally active fragments andvariants of the sequences recited in (a), (b) and (c); and (e) RNAsequences corresponding to the sequences recited in (a), (b), (c) and(d).
 35. A construct including one or more nucleic acids or nucleic acidfragments according to claim
 30. 36. A construct according to claim 35including nucleic acids or nucleic acid fragments encoding both (a) a CSpolypeptide or a CS-like polypeptide and (b) a MDH polypeptide or aMDH-like polypeptide.
 37. A construct according to claim 35 includingnucleic acids or nucleic acid fragments encoding both (a) a CSpolypeptide or a CS-like polypcptide and (b) a PEPC polypeptide or aPEPC-like polypeptide.
 38. A construct according to claim 35 includingnucleic acids or nucleic acid fragments encoding both (a) a MDHpolypcptide or a MDH-like polypeptide and (b) a PEPC polypeptide or aPEPC-like polypeptide.
 39. A construct according to claim 35 includingnucleic acids or nucleic acid fragments encoding all three of (a) a CSpolypeptide or a CS-like polypeptide; (b) a MDH polypeptide or aMDH-like polypeptide; and (c) a PEPC polypeptide or a PEPC-likepolypcptide.
 40. A construct according to claim 35 wherein the one ormore nucleic acids or nucleic acid fragments are operably linked to oneor more regulatory elements such that the one or more nucleic acids ornucleic acid fragments are each expressed.
 41. A construct according toclaim 40, wherein the one or more regulatory elements include a promoterand a terminator, said promoter, nucleic acid or nucleic acid fragmentand terminator being operably linked.
 42. A plant cell, plant, plantseed or other plant part, including a construct according to claim 35.43. A plant, plant seed or other plant part derived from a plant cell orplant according to claim
 42. 44. A method of modifying one or moreselected from the group consisting of organic acid synthesis; organicacid secretion; nutrient acquisition; aluminium and acid soil tolerance;and nitrogen fixation and nodule function; in a plant, said methodincluding introducing into said plant an effective amount of a nucleicacid or nucleic acid fragment according to claim
 30. 45. A methodaccording to claim 44 wherein said method includes introducing into saidplant effective amounts of nucleic acids or nucleic acid fragmentsencoding both (a) a CS polypeptide or CS-like polypeptide and (b) a MDHpolypeptide or MDH-like polypeptide.
 46. A method according to claim 44wherein said method includes introducing into said plant effectiveamounts of nucleic acids or nucleic acid fragments encoding both (a) aCS polypeptide or a CS-like polypeptide and (b) a PEPC polypeptide or aPEPC-like polypeptide
 47. A method according to claim 44 wherein saidmethod includes introducing into said plant effective amounts of nucleicacids or nucleic acid fragments encoding both (a) a MDH polypeptide or aMDH-like polypeptide and (b) a PEPC polypeptide or a PEPC-likepolypeptide.
 48. A method according to claim 44 wherein said methodincludes introducing into said plant effective amounts of nucleic acidsor nucleic acid fragments encoding all three of (a) a CS polypeptide ora CS-like polypeptide; (b) a MDH polypeptide or a MDH-like polypeptide;and (c) a PEPC polypeptide or a PEPC-like polypeptide.
 49. A methodaccording to claim 44 wherein the method is modifying nutrientacquisition and the nutrient is phosphorous.
 50. A substantiallypurified or isolated nucleic acid or nucleic acid fragment including asingle nucleotide polymorphism (SNP) from anucleic acid fragmentaccording to claim
 30. 51. A nucleic acid or nucleic acid fragmentincluding an SNP according to claim 50, wherein said nucleic acid ornucleic acid fragment is from white clover (Trifolium repens) orperennial ryegrass (Lolium perenne).
 52. A substantially purified orisolated polypeptic from a clover (Trifolium), medic (Medicago),ryegrass (Lolium) or fescue (Festuca) species, selected from the groupconsisting of CS and CS-like, MDH and MDH-like and PEPC and PEPC-like;and functionally active fragments and variants thereof.
 53. Apolypeptide according to claim 52, wherein said polypeptide is fromwhite clover (Trifolium repens) or perennial ryegrass (Lolium perenne).54. A polypeptide encoded by a nucleic acid or nucleic acid fragmentaccording to claim
 30. 55. A polypeptide according to claim 52, whereinsaid polypeptide is CS or CS-like and includes an amino acid sequenceselected from the group consisting of sequences shown in SEQ ID NOS 2,12, 18, 20, 328, 337, 350, 352 and 354; and functionally activefragments and variants thereof.
 56. A polypeptide according to claim 52,wherein said polypeptide is MDH or MDH-like and includes an amino acidsequence selected from the group consisting of sequences shown in SEQ IDNOS 22, 31, 35, 37, 39, 41, 45, 47, 112, 114, 116, 184, 186, 206, 219,253, 272, 277, 289, 294, 297, 303, 307 and 309 and functionally activefragments and variants thereof.
 57. A polypeptide according to claim 52,wherein said polypeptide is PEPC or PEPC-like and includes an amino acidsequence selected from the group consisting of sequences shown in SEQ IDNOS 188, 190, 198, 200, 202, 204, 311, 316, 320, 324, 326, and 348; andfunctionally active fragments and variants thereof.